Pulmonary insulin administration using the AERx system: physiological and physicochemical factors influencing insulin effectiveness in healthy fasting subjects.

BACKGROUND: Orally inhaled insulin may provide a convenient and effective therapy for prandial glucose control in patients with diabetes. This study evaluated the influence of formulation pH and concentration and different respiratory maneuvers on pharmacokinetic and pharmacodynamic properties of inhaled insulin. METHODS: Three, open-label crossover studies in a total of 23 healthy subjects were conducted in which the safety, pharmacokinetics, and pharmacodynamics of insulin inhalation were compared to subcutaneous (SC) injection into the abdomen of commercially available regular insulin. A novel, aerosol generating system (AERx Diabetes Management System, Aradigm Corporation, Hayward, CA) was used to deliver aqueous insulin bolus aerosols to the lower respiratory tract from formulations at pH 3.5 or 7.4 and concentrations of U250 (250 U/mL) or U500 (500 U/mL). RESULTS: Time to maximum insulin concentration in serum (Tmax) after SC dosing occurred approximately 50-60 minutes with the time to minimum plasma glucose concentration (i.e., maximum hypoglycemic effect), (TGmin), occurring later, at around 100-120 minutes. In contrast, pulmonary delivery led to a significantly earlier Tmax (7-20 minutes) and TGmin (60-70 minutes), parameters that were shown to be largely unaffected by changing the pH or concentration of the insulin. However, investigation of changes in inhaled volume (achieved by different programming of the AERx system) for administration of the same sized aerosol bolus revealed significant effects. Significantly slower absorption and time to peak hypoglycemic activity occurred when aerosol delivery of insulin occurred during a shallow (approximately 40% vital capacity) as opposed to a deep (approximately 80% vital capacity) inspiration. In addition, it was shown that serum concentration of insulin increased immediately after a series of forced expiraratory maneuvers 30 minutes after inhaled delivery. CONCLUSIONS: Pulmonary delivery of aqueous bolus aerosols of insulin in healthy subjects resulted in rapid absorption with an associated hypoglycemic effect quicker than is achieved after subcutaneous dosing of regular insulin. Inhaled insulin pharmacokinetics and pharmacodynamics were independent of formulation variables (pH, concentration) but affected by certain respiratory maneuvers.

Intraocular pharmacokinetics and safety of a humanized monoclonal antibody in rabbits after intravitreal administration of a solution or a PLGA microsphere formulation.

Poly(lactic-co-glycolic) acid (PLGA) bioresorbable microspheres are used for controlled-release drug delivery and are particularly promising for ocular indications. The objective of the current study was to evaluate the pharmacokinetics and safety of a recombinant human monoclonal antibody (rhuMAb HER2) in rabbits after bolus intravitreal administration of a solution or a PLGA-microsphere formulation. On Day 0, forty-eight male New Zealand white rabbits (2.3-2.6 kg) were immobilized with intramuscular ketamine/xylazine, and the test materials were injected directly into the vitreous compartment. Group 1 animals received rhuMAb HER2 in 50:50 lactide: glycolide PLGA microspheres; Group 2 animals received rhuMAb HER2 in solution (n = 24/group). The dose for each eye was 25 microg (50 microl). After dosing, animals were sacrificed at 2 min, and on 1, 2, 4, 7, 14, 23, 29, 37, 44, 50, and 56 days (n = 2/timepoint/group). Safety assessment included direct ophthalmoscopy, clinical observations, body weight, and hematology and clinical chemistry panels. At necropsy, vitreous and plasma were collected for pharmacokinetics and analysis for antibodies to rhuMAb HER2, and the vitreal pellet (Group 1) was prepared for histologic evaluation. All animals completed the study per protocol-both treatments were well tolerated, and no suppurative or mixed inflammatory cell reaction was observed in the vitreal samples (Group 1) at any of the time points examined. Antibodies to rhuMAb HER2 were detected in plasma samples by Day 7 in both treatment groups, but infrequently in vitreous samples. There were no safety implications associated with this immune response. The in vitro characterization of the PLGA microspheres provided reasonable projections of the in vivo rhuMAb HER2 release kinetics (Group 1). The total amount of antibody that was released was similar in vitro (25.9%) and in vivo (32.4%). RhuMAb HER2 (Group 2) was cleared slowly from the vitreous compartment, with initial and terminal half-lives of 0.9 and 5.6 days, respectively. The volume of distribution approximated the vitreous volume in a rabbit eye.

Comparisons of the intraocular tissue distribution, pharmacokinetics, and safety of 125I-labeled full-length and Fab antibodies in rhesus monkeys following intravitreal administration.

Access of recombinant proteins to the retina following intravitreal administration is poorly understood. A study was conducted in male Rhesus monkeys (15 to 28 mo of age; 2.8-3.3 kg) in order to compare the intraocular tissue distribution, pharmacokinetics, and safety of 125Iodine (I)-labeled full-length humanized rhuMAb HER2 antibody (148 kD) and of 125I-labeled humanized rhuMAb vascular endothelial growth factor Fab antibody (48.3 kD) following bilateral bolus intravitreal injection on day 0 (5 animals/group). The dose administered to each eye was 25 microg (9-10 microCi) in 50 microl. Animals were euthanatized on day 0 (1 hr postdose) and on days 1, 4, 7, and 14. Safety assessment included direct ophthalmoscopy, intraocular pressure measurements, clinical observations, body weight, and hematology and clinical chemistry panels. Blood and vitreous samples were collected daily (blood only) and at necropsy for pharmacokinetics and analysis for antibodies to the test materials; the ocular tissue distribution of the test material was evaluated by microautoradiography. All animals completed the study. Microautoradiography demonstrated that the full-length antibody did not penetrate the inner limiting membrane of the retina at any of the time points examined. In contrast, the Fab antibody fragment diffused through the neural retina to the retinal pigment epithelial layer at the 1-hr time point and persisted in this location for up to 7 days. Systemic exposure to test material was low but variable: the highest plasma concentration of the full-length antibody was 20.3 ng/ml, whereas plasma concentrations for the Fab antibody remained below the limit of quantitation (i.e., <7.8 ng/ml). An immune response to the test material was not evident in either treatment group. The half-life in vitreous was 5.6 days for the full-length antibody and 3.2 days for the Fab antibody. The shorter intravitreal half-life of the Fab antibody is related to its smaller size and its significant diffusion through the retinal layers. The differences in pharmacokinetics and tissue distribution that are noted between the full-length and Fab antibodies in this study identify potential therapeutic approaches that may be exploited in specific disease conditions.

Efficacy and concentration-response of murine anti-VEGF monoclonal antibody in tumor-bearing mice and extrapolation to humans.

The development of a neovascular supply (angiogenesis) is a major aspect of tumorigenesis. Recent work has indicated that vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis. In vitro and in vivo studies have demonstrated that an anti-VEGF antibody is capable of suppressing the growth of human tumor cell lines. The following study was conducted in tumor-bearing nude mice to evaluate the concentration-response relationship of murine anti-VEGF monoclonal antibody (muMAb VEGF) so that an efficacious plasma concentration of the recombinant humanized form (rhuMAb VEGF) in cancer patients could be estimated. (This study was included in our Investigational New Drug application to support the clinical dosing regimen and projected human safety factors for the toxicology program.) Additionally, the growth dynamics of the tumors were evaluated as a function of dose to explore whether a mechanismic interpretation of tumor growth inhibition by muMAb VEGF is possible. On day 1, A673 human rhabdomyosarcoma cells (2 x 10(6) cells/mouse) were injected subcutaneously in 188 beige nude mice (16-24 g). Treatment with muMAb VEGF (0.05-5.0 mg/kg; n = 24/group), phosphate-buffered saline (n = 10), or anti-gp120 isotype-matched control antibody (5.0 mg/kg; n = 10) began 24 hr later. Each animal received intraperitoneal injections of test material twice weekly for 4 wk. Immediately prior to each dose, 2 mice from each muMAb VEGF group were selected randomly, and plasma was collected for pharmacokinetic evaluation; at the end of the study, samples were collected from all animals for pharmacokinetic evaluation. Tumor dimensions were recorded weekly, and at the end of the study, tumor weight and dimensions were recorded. Satisfactory tumor suppression in nude mice was achieved at muMAb VEGF doses of > or =2.5 mg/kg, where the average trough muMAb VEGF plasma concentration was 30 microg/ml (concentrations in individual animals >10 microg/ml). Assuming the pharmacokinetics of rhuMAb VEGF in patients will resemble the pharmacokinetics of a similar humanized anticancer monoclonal antibodies, a clinical dosing regimen was designed to maintain the rhuMAb VEGF plasma concentration in this efficacious range. This study shows an approach that can be used to estimate a human dosing regimen from preclinical pharmacokinetic/pharmacodynamic data. Because we have just initiated clinical trials with rhuMAb VEGF we cannot judge clinical outcome in relation to these preclinical predictions; nonetheless, it is hoped that by sharing our approach and thought processes with other investigators we can assist the discovery and development of anticancer therapeutics.

Pharmacokinetics and interspecies scaling of recombinant human factor VIII.

Recombinant human (rh) factor VIII is a glycoprotein consisting of multiple polypeptides with relative mobilities (M(r)) ranging from 80,000 to 210,000. It is produced in mammalian cells. Single-dose intravenous pharmacokinetic studies were conducted with rh factor VIII (Kogenate rh Antihemophilic Factor, Miles, Inc.) in male mice (21.0-25.8 g) and rats (252.0-254.2 g). Each species received 400 IU/kg, and blood was collected up to 12 hr (mice) or 32.5 hr (rats) post-dose. Immunoreactive factor VIII concentrations in plasma were quantified by a sensitive and specific ELISA. In both species, the disposition profiles were described by the sum of two exponentials. The pharmacokinetics of rh factor VIII in mouse were as follows: clearance, 27.7 ml/hr/kg; initial volume of distribution, 72 ml/kg; steady-state volume of distribution, 148 ml/kg; and terminal half-life, 4.1 hr. In rat, the mean estimates were as follows: clearance, 16.0 ml/hr/kg; initial volume of distribution, 41 ml/kg; steady-state volume of distribution, 125 ml/kg; and terminal half-life, 5.5 hr. These pharmacokinetic parameters for rh factor VIII in animals and human rh factor VIII pharmacokinetic parameters from the literature were evaluated to determine if the parameters can be represented by the allometric relationship, Y = aWb, where Y is the pharmacokinetic parameter, and W is body weight. The following allometric relations were obtained for rh factor VIII: clearance (ml/hr) = 10.4W0.69, half-life (hr) = 7.5 W0.18, initial volume of distribution (ml) = 43.6 W1.04, and steady-state volume of distribution (ml) = 99.1 W0.84. The allometric exponents for each parameter conformed to theory and were within the range of values commonly observed for xenobiotics and therapeutic proteins. These studies suggest that the pharmacokinetics of rh factor VIII in laboratory animals are predictive of the disposition in humans despite the complex nature of its biological interactions and the chemical diversity of the purified material.

[Infectious endocarditis--new aspects of a well-known disease]. / Infekcná endokarditída--nové pohl'ady na známe ochorenie.

The authors discuss the incidence, etiology and pathogenesis of infectious endocarditis. They mention the incidence and therapeutic results in patients with infectious endocarditis hospitalized at the First Medical Clinic in Martin in 1988-1993. In the conclusion they present general principles of the treatment of infectious endocarditis.

What dual-action agents reveal about acid secretion: a combined experimental and modeling analysis.

A previously described simple mathematical model for gastric acid secretion is employed to characterize the acid secretion response to thiocyanate and omeprazole, two partially conservative inhibitors that inhibit both the formation and translocation of acid. In addition, the effect of dithiothreitol on the omeprazole inhibition was investigated. The model parameters were estimated from four data sets by a non-linear least squares procedure: a dynamic (time-dependent) set, made up of individual acid secretion rate curves; and three integral (time-independent) sets consisting of the curves of acid secreted and suppressed above and below baseline, respectively, and the index of conservation, all three as functions of agent exposure. At the translocation step the inhibition function was the same for the two inhibitors, and though similar at the formation step, they differ in that a Hill coefficient larger than unity is necessary in the thiocyanate inhibition function of the acid formation. Dithiothreitol protects only the formation step from inhibition by omeprazole. Hence, the binding sites for omeprazole are different for formation and translocation. This study exemplifies a non-invasive method for the investigation of the mechanism of gastric acid secretion following perturbation by external agents.

Dynamics of a metabolic system: what single-action agents reveal about acid secretion.

A simple single-state nonlinear mathematical model of an open metabolic system is shown to be an adequate representation of acid secretion in frog gastric mucosa. The parameters of the elemental model were estimated from data, and subsystems of augmented models were established for histamine, a nonconservative stimulus acting via binding to receptors, and for two inhibitors of acid secretion. The latter included metiamide, a nonconservative histamine antagonist, which affects the formation of acid by competitive binding to the histamine receptors, and nitrite, a conservative inhibitor, which affects the rate of acid translocation. For parameter estimation, two data sets were analyzed by a nonlinear least-squares procedure: a dynamic (time dependent) set consisting of individual acid secretion rate curves and an integral (time independent) set consisting of the curves of acid secreted and suppressed above or below baseline, respectively, as functions of agent exposure. Because the model is instrumental in the estimation of parameters of subsystems that are not accessible through direct observation, it can serve as a research tool in the investigation of the mechanism of gastric acid secretion under a variety of experimental conditions.

Plasma protein osmotic pressure equations and nomogram for sheep.

The equations developed by Landis and Pappenheimer (Handbook of Physiology. Circulation, 1963, p. 961-1034) for calculating the protein osmotic pressure of human plasma proteins have been frequently used for other animal species without regard to the fractional albumin concentration or correction for protein-protein interaction. Using an electronic osmometer, we remeasured the protein osmotic pressure of purified sheep albumin and sheep plasma partially depleted of albumin. We measured protein osmotic pressures of serial dilutions over the concentration range 0-180 g/l for albumin and 0-100 g/l for the albumin-depleted proteins at room temperature (26 degrees C). Using a nonlinear least squares parameter-fitting computer program, we obtained the equation of best fit for purified albumin, and then we used that equation together with the measured albumin fraction to obtain the best-fit equation for the nonalbumin proteins. The equation for albumin is IIcmH2O,39 degrees C = 0.382C + 0.0028C2 + 0.000013C3, where C is albumin concentration in g/l. The equation for the nonalbumin fraction is IIcmH2O,39 degrees C = 0.119C + 0.0016C2. Up to 200- and 100-g/l protein concentration, respectively, these equations give the least standard error of the estimate for each of the virial coefficients. The computed number-average molecular weight for the nonalbumin proteins is 222,000. Using the new equations, we constructed a nomogram, based on the one of Nitta and co-workers (Tohoku J. Exp. Med. 135: 43-49, 1981). We tested the nomogram using 144 random samples of sheep plasma and lymph from 31 sheep. We obtained a correlation coefficient of 0.99 between the measured and nomogram estimates of protein osmotic pressure.

Extracellular potassium is necessary for acid translocation but not for acid formation in gastric mucosa.

Removal of potassium from the nutrient solution of a gastric mucosa results in monotonic decline in acid secretion rate. When potassium is added back to the nutrient solution, acid secretion recovers. In both spontaneously and sub-maximally secreting tissue the recovery of the acid secretion rate takes the form of a transient overshoot above the baseline such that the amount of suppressed acid in the absence of potassium is equal to the amount of acid that is secreted above baseline once potassium is added back. The index of conservation r is zero, i.e., the effect is conservative. In maximally secreting tissue the secretion rate only returns to pre-inhibitory level without an overshoot. The net effect is nonconservative with r = -1. Conservative effect under maximally stimulating conditions was unmasked by exposing the tissue to a stimulus for a specific length of time and comparing amount of acid secreted when nutrient potassium was present with amount of acid secreted when potassium was removed and then added back to the nutrient solution. Stimulation with forskolin is not expressed in the absence of potassium but is unmasked once potassium is added back to the nutrient solution. The conclusion is: removing potassium from the nutrient side inhibits proton translocation by decreasing Vmax and/or increasing Km but is without effect on the formation of acid. Adding potassium back restores the parameter(s) for the translocation step.

[The effect of quinidine on digoxin plasma levels after discontinuation of digoxin therapy]. / Vplyv chinidínu na plazmaticku hladinu digoxínu po vynechaní digoxínovej liecby.

The authors submit a report on the effect of quinidine on plasma digoxin. The investigation was made in 20 patients wit supraventricular dysrhythmia. After initial digoxin treatment they proceeded with quinidine six hours after the last digoxin dose. Although they did not administer digoxin, the digoxin level increased during quinidine treatment in the course of 12 hours from 2.4 +/- 1.6 ng/ml to 3.8 +/- 12.4 ng/ml. The authors draw attention to the importance of following up digoxin levels in the mentioned drug combination which is supposed to prevent serious complication caused by possible digoxin intoxication.

Accumulation of weak base in gastric mucosa provides evidence for an acidic storage compartment.

Uptake and release of acridine orange (AO), a fluorescent weak base that accumulates in acidic spaces, were studied in perfused frog gastric mucosa. Tissue was mounted between two flow-through chambers and loaded with AO on the mucosal side. AO washout and acid secretion rate were monitored simultaneously by a flow-through fluorescence detector and a pH-stat, respectively. Data were displayed on a computer screen, stored, and analyzed. AO, in concentrations as high as 0.02 mM, does not affect the acid secretion rate. Nonlinear least-squares analysis of AO washout curves resolved two exponential components: a faster component associated mainly with AO washout from the chamber and a slower component reflecting primarily AO washout from the tissue. The slower exponential declines more slowly at higher concentrations and/or longer duration of AO loading, whereas the faster exponential is unaffected. AO washout is unaffected by the level of the steady-state acid secretion rate. Nitrite inhibits the acid secretion rate but does not affect the AO washout. When nitrite is removed, acid secretion rate and fluorescence (AO concentration in the mucosal medium) increase simultaneously and transiently. The net amount of AO released from the tissue is proportional to the net amount of acid released. Stimulation by secretagogue in basally secreting tissue causes synchronous transient increases in acid secretion rate and fluorescence. We conclude that accumulation of AO provides evidence for the existence of an intracellular storage pool of free protons within the transporting epithelium.

The hepatocellular transport of sulfobromophthalein-glutathione by clofibrate treated, perfused rat liver.

The hypolipidemic drug clofibrate is known to affect the hepatic transport of various organic anions including bilirubin, fatty acids and sulfobromophthalein. Changes in the rate of metabolism and/or intracellular transport have been claimed responsible for the effect. To evaluate these possibilities, the transport of sulfobromophthalein-glutathione, a model compound that does not require metabolism for biliary excretion, was studied in perfused livers isolated from clofibrate-treated and control rats. Cytosolic fatty acid binding protein and glutathione S-transferase activity were also measured. Clofibrate treatment significantly increased liver weight; as a result glutathione S-transferase activity (toward 1-chloro-2,4-dinitrobenzene) fell if expressed per gram of liver (4560 +/- 420 (SE) vs 7010 +/- 260 nmoles/min for clofibrate treated and controls respectively, p less than 0.002), but was unchanged when expressed per total liver (60.8 +/- 6.5 vs 64.6 +/- 3.5 mumoles/min for clofibrate and controls p greater than 0.5). Irrespective of how it was expressed fatty acid binding protein was significantly increased by the drug treatment. Steady state sulfobromophthalein-glutathione removal velocity was saturable with increasing concentrations of sulfobromophthalein-glutathione in both control and clofibrate-treated livers. Steady state extraction ratio, as well as Vmax and Km for removal, did not differ between the two groups. In keeping with other observations, these data collectively indicate that the hepatic steady state removal of nonmetabolized compounds is not affected by clofibrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Sex differences in sulfobromophthalein-glutathione transport by perfused rat liver.

Sex differences have been described in the hepatic transport of many organic anions. Proposed mechanisms include differences in the rate of metabolism, in the degree of binding to cytoplasmic proteins, and in the rate of membrane transport. To better define these factors, we used the perfused rat liver to study the hepatic transport of the glutathione conjugate of sulfobromophthalein (BSP-GSP), a model compound that does not require metabolism for excretion. Hepatic transport of BSP-GSH was saturable for both sexes. Clearance of BSP-GSH from 1% albumin solutions at steady-state was 35-52% greater in female livers than in male livers, and reflected a 47% larger apparent Vmax with no change in the apparent Km. Analysis of the rate of disappearance of BSP-GSH from recirculating perfusate and its appearance in bile using a simple two-compartment model indicated that the ratio of influx to efflux was greater in female livers. These findings are compatible with sex-related differences in the electrochemical driving forces for BSP-GSH uptake.

Phenobarbital specifically increases the hepatocellular uptake of sulfobromophthalein-glutathione.

The transport of sulfobromophthalein glutathione was studied in perfused livers isolated from phenobarbital treated and control rats. Phenobarbital increased the cell size and the uptake of sulfobromophthalein glutathione. The effect on uptake is specific since in phenobarbital treated livers each unit of hepatocyte surface area takes up more sulfobromophthalein glutathione than controls. The cellular hypertrophy does not involve all cell functions; total and specific content of cytosolic fatty acid binding protein for example, were unchanged by phenobarbital. The increase in Vmax and influx rate constant for sulfobromophthalein glutathione uptake suggest that phenobarbital increases the amount of membrane carriers or their rate of cycling.

Conservative and nonconservative inhibitors of gastric acid secretion.

Inhibitors of the initial step (H2-antagonist) and of the final step (thiocyanate, SCN-; and nitrite, NO2-) were used to study the dynamics of acid secretion in isolated frog gastric mucosa. Tissues were mounted in flow-through chambers, and the acid secretion rate (SR) was recorded on a pH-stat microprocessor. Continuous presence of H2-antagonist decreases the SR to a lower steady state, and on removal the SR returns to basal SR, causing a net loss of acid, the nonconservative effect. The amount of lost acid is a unique function of exposure, thus, independent of the patterns (pulses or steps) of inhibition. In contrast, continuous presence of SCN- or NO2- (below 3 mM) results in an undershoot in SR with a return to basal SR, whereas at higher concentrations there is no return. Removal of these inhibitors causes an overshoot in SR with return to basal SR. The rebound acid is equal to acid suppressed by NO2- and low concentration of SCN-, resulting in no net loss of acid, the conservative effect, whereas at high concentrations of SCN- there is an apparent loss of acid. In maximally secreting tissue the overshoot of SR is not observed. However, the acid is not lost, merely delayed. In resting tissue NO2- also merely delays the exit of the acid produced in response to forskolin. The rebound acid is proposed to reside in a sequestered "acid" pool that is stable for at least 120 min. Results with NO2- and SCN- suggest an effect on a saturable exit enzyme, possibly the K+-H+-ATPase.

Comparing binding of histamine and H2-antagonist with their effects on gastric acid secretion.

A microsomal fraction from isolated frog gastric mucosa was used to study the binding of labeled histamine, labeled metiamide (a histamine H2-antagonist), and competition between labeled histamine and unlabeled metiamide. The separation of free from bound ligand was done by gel chromatography. The acid secretion was studied in frog gastric mucosa in vitro by a pH-stat method. The binding data could be interpreted in terms of two independent binding sites for both histamine and metiamide. However, the competition between histamine and metiamide does not support the independence of the sites. Moreover, the dissociation kinetics of labeled metiamide in the presence of unlabeled metiamide is non-monotone and, thus, indicates cooperativity. In the physiological studies, the dependence of the rate of acid secretion on histamine stimulation occurs within very narrow limits, which is the result of characteristics other than related to binding. However, the total amount of acid secreted caused by a pulse of histamine does indicate two sites, of which the high-affinity site is the more effective. Metiamide inhibition of acid secretion can be interpreted as an interaction between high-affinity sites of histamine and metiamide. Overall, studies involving physiological effects provide less precise data than the direct binding studies.

Fluid phase endocytosis by cultured rat hepatocytes and perfused rat liver: implications for plasma membrane turnover and vesicular trafficking of fluid phase markers.

Hepatocytes take up a variety of ligands via receptor-mediated endocytosis, yet little is known regarding either the volume of fluid or the amount of membrane internalized via endocytosis in liver cells. In these studies, we have utilized radiolabeled inulin to characterize fluid phase endocytosis by rat hepatocytes in primary culture and perfused rat liver. Uptake of inulin by cultured hepatocytes was nonlinear with time, occurring most rapidly during the first 2 min. Inulin uptake and efflux in cultured hepatocytes and inulin uptake by perfused rat liver were kinetically compatible with the entry of inulin into a rapidly (t1/2, 1-2 min) turning-over (presumably endosomal) compartment that exchanged contents with the extracellular space and comprised approximately 3% of hepatocyte volume, as well as entry into and concentration of inulin within slowly (t1/2, greater than 1 hr) turning-over storage compartments. Based on inulin uptake, it is estimated that cultured hepatocytes endocytosed the equivalent of 20% or more of their volume and 5 or more times their plasma membrane surface area each hour. Neither chloroquine (1 mM) nor taurocholate (200 microM) affected inulin handling by cultured cells, whereas colchicine (10 microM) inhibited transfer to storage compartments by greater than 50%. In conjunction with our previous observations, the present findings suggest that inulin endocytosed across the basolateral membrane is largely (congruent to 80%) regurgitated back into plasma, with smaller amounts transported to intracellular storage compartments (congruent to 18%) or to bile (congruent to 2%). Transport of inulin via these pathways is unaffected by taurocholate and does not require vesicle acidification, whereas intact microtubular function is required for transfer to storage compartments or biliary secretion.

Asynchronies of diphenhydramine plasma-performance relationships.

The relation between performance on driving-related tasks and plasma levels of diphenhydramine was studied in eight male volunteers over 24 hours following oral administration. DPH plasma concentrations rose to peak levels in 1.5-2.5 hours varying with dose, declining to a nearly constant level by 12-24 hours. For all behavioral measures, the shape of performance curves over time was similar to that of plasma, reaching maximum decrements in 1-4 hours. The relation between plasma levels and performance was asynchronous varying with behavioral measure and dose. The use of plasma DPH values to predict performance decrements is limited due to the bivalued nature of these relationships in time. Nonetheless, it would appear that if DPH is administered in the therapeutic dosage range at the intervals typically recommended for cold symptoms and allergies, it appears some aspects of human performance may be impaired.

Biliary secretion of fluid-phase markers by the isolated perfused rat liver. Role of transcellular vesicular transport.

In these studies, we have used several approaches to systematically explore the contribution of transcellular vesicular transport (transcytosis) to the blood-to-bile movement of inert fluid-phase markers of widely varying molecular weight. First, under steady-state conditions, the perfused rat liver secreted even large markers in appreciable amounts. The bile-to-plasma (B/P) ratio of these different markers, including microperoxidase (B/P ratio = 0.06; mol wt = 1,879), insulin (B/P ratio = 0.09, mol wt = 5,000), horseradish peroxidase (B/P ratio = 0.04, mol wt = 40,000), and dextran (B/P ratio = 0.09, mol wt = 70,000), exhibited no clear ordering based on size alone, and when dextrans of two different sizes (40,000 and 70,000 mol wt) were studied simultaneously, the relative amounts of the two dextran species in bile were the same as in perfusate. Taurocholate administration produced a 71% increase in bile flow but little or no (0-20%) increase in the output of horseradish peroxidase, microperoxidase, inulin, and dextran. Second, under nonsteady-state conditions in which the appearance in or disappearance from bile of selected markers was studied after their abrupt addition to or removal from perfusate, erythritol reached a B/P ratio of 1 within 2 min. Microperoxidase and dextran appeared in bile only after a lag period of approximately 12 min and then slowly approached maximal values, whereas sucrose exhibited kinetically intermediate behavior. A similar pattern was observed after removal of greater than 95% of the marker from the perfusate. Erythritol rapidly reapproached a B/P ratio of 1, whereas the B/P ratio for sucrose, dextran, and microperoxidase fell much more slowly and exceeded 1 for a full 30 min after perfusate washout. Finally, electron microscopy and fluorescence microscopy of cultured hepatocytes demonstrated the presence of horseradish peroxidase and fluorescein-dextran, respectively, in intracellular vesicles, and fractionation of perfused liver homogenates revealed that at least 35-50% of sucrose, inulin, and dextran was associated with subcellular organelles. Collectively, these observations are most compatible with a transcytosis pathway that contributes minimally to the secretion of erythritol, but accounts for a substantial fraction of sucrose secretion and virtually all (greater than 95%) of the blood-to-bile transport of microperoxidase and larger markers. These findings have important implications with respect to current concepts of canalicular bile formation as well as with respect to the conventional use of solutes such as sucrose as markers of canalicular or paracellular pathway permeability.